The initiation of DNA synthesis and its regulation is a fundamental process of biology that impacts virtually every aspect of human health. Proper replication determines the fate of cells during early development and throughout adult life. DNA polymerases cannot synthesize DNA without a primer, and primase is the specialized RNA polymerase capable of de novo synthesis of short RNA primers during replication. In eukaryotes, primase functions within a heterotetrameric primase-DNA polymerase alpha (pol alpha) complex. This complex is uniquely capable of switching from the synthesis of RNA by primase to the synthesis of DNA by pol alpha. The synthesized RNA-DNA primer is required for further DNA synthesis by the major replicative DNA polymerases. In humans, the primase component consists of a small catalytic subunit (p49) and a large subunit (p58), and pol alpha is comprised of a catalytic subunit (p180) and an accessory subunit B (p70). The concerted actions of primase and pol alpha are critical for accurate genome duplication. Malfunction of primase-pol alpha complex causes global genome instability and is linked to the onset and progression of cancer and other diseases. Currently, the details for primase-pol alpha complex organization and function, including the mechanisms of unit size RNA primer synthesis and subsequent internal transfer to pol alpha are very limited. The goal of our project is to determine the structural basis of human primase-pol alpha complex function and reveal the biological consequences of alterations in this complex. To achieve our goal we will determine the mechanism of unit-length RNA primer synthesis and counting by human primase (Aim 1), the structural and functional consequences of primase integration into the pol alpha complex (Aim 2), and the mechanism of substrate switch from primase to pol alpha (Aim 3). Our studies will involve a variety of methods: X-ray crystallography, small angle X-ray scattering (SAXS), surface plasmon resonance (SPR), single molecule experiments, yeast two-hybrid and polymerase reactions assays. We also will examine the in vivo impact of mutations affecting primase-pol alpha activities on genome stability in a yeast model system.